Abstract
Apoptotic markers consist of either caspase substrate cleavage products or phenotypic changes that manifest themselves as a consequence of caspase-mediated substrate cleavage. We have shown recently that pharmacological inhibitors of caspase activity prevent the appearance of two such apoptotic manifestations, alphaII-spectrin cleavage and DNA fragmentation, but that blockade of the latter required a significantly higher concentration of inhibitor. We investigated this phenomenon through the use of a novel radiolabeled caspase inhibitor, [(125)I]M808, which acts as a caspase active site probe. [(125)I]M808 bound to active caspases irreversibly and with high sensitivity in apoptotic cell extracts, in tissue extracts from several commonly used animal models of cellular injury, and in living cells. Moreover, [(125)I]M808 detected active caspases in septic mice when injected intravenously. Using this caspase probe, an active site occupancy assay was developed and used to measure the fractional inhibition required to block apoptosis-induced DNA fragmentation. In thymocytes, occupancy of up to 40% of caspase active sites had no effect on DNA fragmentation, whereas inhibition of half of the DNA cleaving activity required between 65 and 75% of active site occupancy. These results suggest that a high and persistent fractional inhibition will be required for successful caspase inhibition-based therapies.
Highlights
The deliberate removal of excess or damaged cells is a universal feature of multicellular organisms and proceeds by a biochemical suicide program known as apoptosis
We investigated this phenomenon through the use of a novel radiolabeled caspase inhibitor, [125I]M808, which acts as a caspase active site probe. [125I]M808 bound to active caspases irreversibly and with high sensitivity in apoptotic cell extracts, in tissue extracts from several commonly used animal models of cellular injury, and in living cells
We compared caspase active site occupancy with DNA fragmentation, and demonstrate that inhibition of up to 40% of caspase activity has no effect on apoptosis-induced DNA fragmentation, suggesting that a high fractional inhibition of caspases is required for inhibition of DNA cleavage
Summary
We compared caspase active site occupancy with DNA fragmentation, and demonstrate that inhibition of up to 40% of caspase activity has no effect on apoptosis-induced DNA fragmentation, suggesting that a high fractional inhibition of caspases is required for inhibition of DNA cleavage. The caspase active site probe was able to detect active caspases in vivo, its uneven distribution precluded determination of caspase active site occupancy. We devised an ex vivo method that estimates the percentage of caspase that must be blocked to inhibit DNA cleavage in vivo, and applied the methodology to the rat cecal ligation and puncture (CLP) sepsis model
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